The reactive nitrogen species and alkylating agents generated endogenously and exogenously are the major cause of cellular genotoxicity, which is thought to cause multiple diseases such as cancer and aging. Such alkylated and deaminated base lesions are generally repaired via the base excision repair (BER) pathway, initiated when a DNA glycosylase removes the damaged base. Among these, a series of structurally diverse damaged purines are repaired by N-methylpurine DNA-glycosylase (MPG), present in all species from bacteria to man. Although 3D structure of human(h) MPG has recently been solved by X-ray crystallography, little is known about its detailed mechanisms for recognition of substrates with various structures, or for subsequent catalysis. Our preliminary results show that AP-endonuclease interacts with and stimulates MPG. Moreover, our novel preliminary observations suggesting that MPG is a component of long patch BER complex provide the ground work to test our central hypothesis that the interaction with cellular factors including other BER proteins or post-translational modification may select the BER subpathways (short or long patch) and modulate the activity of MPG to approximate the catalytic rates of its bacterial counterpart depending on cellular necessity. Our specific aims are to: (1) determine the molecular mechanisms of substrate recognition and catalysis by MPG by isolating a series of site-specific MPG mutants and testing for their activities in non-target, mismatched, substrate DNA binding and bending as well as base-flipping and catalysis; (2) elucidate the effect of BER or other cellular proteins on the enzymatic activity of MPG by testing protein-protein interactions and detailed kinetic analysis; and (3) elucidate the in vivo status of short and long patch base excision repair pathways of alkylated and deaminated bases by developing a novel plasmid based in vivo assay. Our long-term goal is comprehensive understanding of the role and regulation of MPG as a component of mammalian BER system for repair of cellular alkylation/deamination damage. This knowledge will allow us eventually to devise strategies for modulating MPG expression for chemopreventive and therapeutic purposes.